Abstract
At present, there are no effective antifibrotic drugs for patients with chronic liver disease; hence, the development of antifibrotic therapies is urgently needed. Here, we performed an experimental and translational study to investigate the potential and underlying mechanism of quercetin treatment in liver fibrosis, mainly focusing on the impact of quercetin on macrophages activation and polarization. BALB/c mice were induced liver fibrosis by carbon tetrachloride (CCl4) for 8 weeks and concomitantly treated with quercetin (50 mg/kg) or vehicle by daily gavage. Liver inflammation, fibrosis, and hepatic stellate cells (HSCs) activation were examined. Moreover, massive macrophages accumulation, M1 macrophages and their related markers, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and monocyte chemotactic protein-1 (MCP-1) in livers were analyzed. In vitro, we used Raw 264.7 cells to examine the effect of quercetin on M1-polarized macrophages activation. Our results showed that quercetin dramatically ameliorated liver inflammation, fibrosis, and inhibited HSCs activation. These results were attributed to the reductive recruitment of macrophages (F4/80+ and CD68+) into the liver in quercetin-treated fibrotic mice confirmed by immunostaining and expression levels of marker molecules. Importantly, quercetin strongly inhibited M1 polarization and M1-related inflammatory cytokines in fibrotic livers when compared with vehicle-treated mice. In vitro, studies further revealed that quercetin efficiently inhibited macrophages activation and M1 polarization, as well as decreased the mRNA expression of M1 macrophage markers such as TNF-α, IL-1β, IL-6, and nitric oxide synthase 2. Mechanistically, the inhibition of M1 macrophages by quercetin was associated with the decreased levels of Notch1 expression on macrophages both in vivo and in vitro. Taken together, our data indicated that quercetin attenuated CCl4-induced liver inflammation and fibrosis in mice through inhibiting macrophages infiltration and modulating M1 macrophages polarization via targeting Notch1 pathway. Hence, quercetin holds promise as potential therapeutic agent for human fibrotic liver disease.
Highlights
Collagen deposition in the liver of CCl4-treated mice was confirmed by computerized image analysis of the fibrotic area, whereas fibrotic mice treated with quercetin markedly attenuated the progression of CCl4-induced fibrosis when compared with vehicle-treated fibrotic mice (4.93 vs. 11.22%, P < 0.001; Figure 1E)
We found that chronic CCl4 injection increased M1 macrophages, as determined by IHC staining with antibodies against CD11c, IL-12, and IRF5 (Figure 5A) and by quantification of CD11c+ cells, IL-12+ cells, and interferon-regulatory factor (IRF)-5+ cells in liver sections (Figure 5B), while these positive cells were restricted to liver sinusoids and fibrotic septa in CCl4-induced fibrotic mice, and barely detectable in healthy livers; quercetin-treated CCl4 mice suppressed M1 macrophages polarization as confirmed by M1-associated markers (Figures 5A,B)
We used chitinase-3-like 3 (Chi3l3; known as Ym-1; mouse only) and CD163 (Beljaars et al, 2014; Alzaid et al, 2016) as molecular M2 macrophage markers; and our results demonstrated that the expression of M2 markers in livers was clearly higher after chronic CCl4 damage as demonstrated by immunostaining with antibodies against Ym-1 and CD163 (Figure 6A); mice given quercetin decreased those M2 macrophages staining signaling when compared with the vehicletreated mice (Figure 6A)
Summary
Liver fibrosis is a typical wound-healing process triggered by liver injury and inflammation resulting from a wide variety of etiologies, such as chronic virus infection (mainly hepatitis B and C viruses), alcoholic and nonalcoholic steatohepatitis (NASH), drugs, cholestasis, and autoimmune hepatitis (Friedman, 2008; Pellicoro et al, 2014; Tsuchida and Friedman, 2017). It has been generally accepted that resident hepatic stellate cells (HSCs), which become activated and transdifferentiate into myofibroblast-like cells in response to chronic liver injury, are the major source of ECM during the process of liver fibrogenesis (Pellicoro et al, 2014; Seki and Schwabe, 2015; Tsuchida and Friedman, 2017). It has become evident that HSCs activation results from the inflammatory activity of liver immune cells, predominantly macrophages (Pellicoro et al, 2014; Seki and Schwabe, 2015; Li et al, 2017b). Given the critical regulatory role of macrophages in HSCs activation and liver fibrosis, we believe that it provides therapeutic targets promising application in the future
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